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Chapter 19 : Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production
Authors:
Michael E. Pyne,
Karan S. Sukhija,
C. Perry Chou
Content Type: Reference
Publication Year:
2010
Category: Applied and Industrial Microbiology
Book DOI:
10.1128/9781555816827
Chapter DOI:
10.1128/9781555816827.ch19
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Abstract:
Typically, the biotechnological basis for strain improvement to enhance recombinant protein production relies on the permanent implementation of desirable traits into the production strain to stimulate both cell growth and functional expression of the target gene during the cultivation. This chapter reviews the major technical issues associated with Escherichia coli strain engineering to enhance recombinant protein production and directs the reader to protocols appropriate for specific applications. Theoretically, strategies based on enhancing the limiting step can lead to an overall improvement in recombinant protein production. Stationary-phase genes encode proteins that may lead to a reduction in cellular and metabolic activity, which can negatively affect recombinant protein production, and as such, these genes are targets for strain improvement. The recently commercialized recombineering protocol from Gene Bridges, also based on λ Redmediated recombination, allows versatile chromosomal engineering, including gene disruption, deletion, insertion, point mutation, modification, and even promoter fine-tuning, and can serve as a versatile manipulation tool for strain improvement and even optimization. The general guidelines for strain improvement are (i) to ensure the genetic stability of the host/vector system, (ii) to maximize the synthesis fluxes for all the gene expression steps (i.e., transcription, translation, and posttranslational processing steps), (iii) to ensure the flux balance of these protein synthesis steps, (iv) to stabilize all the expression intermediates and final products, and (v) to minimize the physiological impact associated with high-level gene expression and high-cell density cultivation.
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
Figures
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production
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Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19fig01
FIGURE 1
Flowchart for E. coli strain improvement to enhance recombinant protein production. The strategies depend on identification of the specific factors limiting the overall recombinant protein yield and include optimization of the host/vector system, expression variables, and expression sequence. Refer to
Table 1
for a list of technical limitations and how to deal with them. DO, dissolved oxygen.
10.1128/9781555816827/f0275-01_thmb.gif
10.1128/9781555816827/f0275-01.gif
FIGURE 1
Flowchart for E. coli strain improvement to enhance recombinant protein production. The strategies depend on identification of the specific factors limiting the overall recombinant protein yield and include optimization of the host/vector system, expression variables, and expression sequence. Refer to Table 1 for a list of technical limitations and how to deal with them. DO, dissolved oxygen.
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19fig02
FIGURE 2
Molecular events associated with recombinant protein production in E. coli. Recom-binant protein production involves a series of complex molecular mechanisms, such as replication of the expression vector, transcription and translation of the gene of interest, and various post-translational processing steps (including protein secretion, folding, and disulfide bond formation). Production can be limited by low efficiency at any one of these steps or by an abnormal event that diverts protein synthesis into a nonproductive pathway (e.g., protein misfolding or degradation of DNA, mRNA, or protein).
10.1128/9781555816827/f0276-01_thmb.gif
10.1128/9781555816827/f0276-01.gif
FIGURE 2
Molecular events associated with recombinant protein production in E. coli. Recom-binant protein production involves a series of complex molecular mechanisms, such as replication of the expression vector, transcription and translation of the gene of interest, and various post-translational processing steps (including protein secretion, folding, and disulfide bond formation). Production can be limited by low efficiency at any one of these steps or by an abnormal event that diverts protein synthesis into a nonproductive pathway (e.g., protein misfolding or degradation of DNA, mRNA, or protein).
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19fig03
FIGURE 3
A typical E. coli expression vector (i.e., plasmid) for recombinant protein production. Several expression and cloning features are shown, including Reg (gene encoding the regulator, either transcriptional activator or repressor), P (promoter), O (operator), rbs (ribosome binding site), SP (signal peptide), N-tag (N-terminal fusion tag), C-tag (C-terminal fusion tag), T (terminator), CS (cloning site), MCS (multiple cloning sites), Ori (replication origin), DRUG (drug resistance gene), ATG (initiation codon encoding methionine), P-ase (protease cleavage site), and End (stop codon). Note that, depending on the cloning site(s) for insertion of a target gene (i.e., open reading frame [ORF]), a transcriptional or translational fusion vector can be constructed to express a gene product (either ORF or ORF-fusion) containing various feature domains and targeting in the cytoplasm or extracytoplasmic compartment.
10.1128/9781555816827/f0276-02_thmb.gif
10.1128/9781555816827/f0276-02.gif
FIGURE 3
A typical E. coli expression vector (i.e., plasmid) for recombinant protein production. Several expression and cloning features are shown, including Reg (gene encoding the regulator, either transcriptional activator or repressor), P (promoter), O (operator), rbs (ribosome binding site), SP (signal peptide), N-tag (N-terminal fusion tag), C-tag (C-terminal fusion tag), T (terminator), CS (cloning site), MCS (multiple cloning sites), Ori (replication origin), DRUG (drug resistance gene), ATG (initiation codon encoding methionine), P-ase (protease cleavage site), and End (stop codon). Note that, depending on the cloning site(s) for insertion of a target gene (i.e., open reading frame [ORF]), a transcriptional or translational fusion vector can be constructed to express a gene product (either ORF or ORF-fusion) containing various feature domains and targeting in the cytoplasm or extracytoplasmic compartment.
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19fig04
FIGURE 4
Chromosomal engineering of E. coli based on homologous recombination for either site-specific gene knockout or gene insertion. The target allele/site is first selected and the exogenous segment is prepared in vitro (e.g., by PCR). Because E. coli is artificially transformable, the exogenous DNA can be delivered into the recipient cell through electroporation. The efficiency of in vivo recombination can be enhanced by expressing key enzyme(s) associated with the recombination. A drug resistance marker is often introduced as the major replacing cassette or cotransduced with a new gene for selection of transformed cells, and can be subsequently deleted in vivo (e.g., by FLP recombination).
10.1128/9781555816827/f0281-01_thmb.gif
10.1128/9781555816827/f0281-01.gif
FIGURE 4
Chromosomal engineering of E. coli based on homologous recombination for either site-specific gene knockout or gene insertion. The target allele/site is first selected and the exogenous segment is prepared in vitro (e.g., by PCR). Because E. coli is artificially transformable, the exogenous DNA can be delivered into the recipient cell through electroporation. The efficiency of in vivo recombination can be enhanced by expressing key enzyme(s) associated with the recombination. A drug resistance marker is often introduced as the major replacing cassette or cotransduced with a new gene for selection of transformed cells, and can be subsequently deleted in vivo (e.g., by FLP recombination).
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19fig05
FIGURE 5
Chromosomal engineering of E. coli based on the intron gene-targeting system for either site-specific gene knockout or gene insertion. The target allele/site is first selected and the gene sequence is entered into the EcI5 computer algorithm to obtain putative insertion sites and corresponding mutagenesis primers. The intron is then retargeted, ligated into a targetron vector, and expressed within the appropriate host strain. The pACD3-EcI5 vectors contain a convenient MluI restriction site for inserting cargo genes such as a drug marker for knockout selection or a foreign gene for chromosomal expression in E. coli.
10.1128/9781555816827/f0282-01_thmb.gif
10.1128/9781555816827/f0282-01.gif
FIGURE 5
Chromosomal engineering of E. coli based on the intron gene-targeting system for either site-specific gene knockout or gene insertion. The target allele/site is first selected and the gene sequence is entered into the EcI5 computer algorithm to obtain putative insertion sites and corresponding mutagenesis primers. The intron is then retargeted, ligated into a targetron vector, and expressed within the appropriate host strain. The pACD3-EcI5 vectors contain a convenient MluI restriction site for inserting cargo genes such as a drug marker for knockout selection or a foreign gene for chromosomal expression in E. coli.
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
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Tables
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production
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Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19tab01
TABLE 1
Factors limiting recombinant protein production in E. coli and corresponding strategies for overcoming these limitations
TABLE 1
Factors limiting recombinant protein production in E. coli and corresponding strategies for overcoming these limitations
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19
/content/10.1128/9781555816827.ch19.ch19tab02
TABLE 2
Genetic elements found in a typical expression vector used for recombinant protein production
TABLE 2
Genetic elements found in a typical expression vector used for recombinant protein production
Citation:
Pyne M, Sukhija K, Chou C.
2010.
Strain Improvement of Escherichia coli To Enhance Recombinant Protein Production,
p 273-286.
In
Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed),
Manual of Industrial Microbiology and Biotechnology, Third Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555816827.ch19